Botulinum toxin targets the neuromuscular junction (NMJ) producing the fatal paralytic disease botulism. Environmental exposure occurs primarily from contaminated foodstuffs, or from contaminated soil. Exposure to botulinum toxin can also occur through inhalation of aerosolized toxin used as a biological weapon. There are no effective treatment measures for botulism once symptoms appear, and death occurs due to respiratory muscle paralysis. Ironically, botulinum toxin is also a valued drug used to treat neuromuscular diseases characterized by muscle spasticity. Our long range goal is to understand how this toxin selectively targets cholinergic nerve terminals of NMJs, in order to reduce its risk to human health and improve its clinical utility. The objective of this particular application is to define the molecular targets on the nerve terminal membrane that are responsible for the selective action of botulinum toxin serotypes A, B and E at the mammalian NMJ. Substantial evidence indicates that productive binding of botulinum toxins requires both polysialogangliosides and glycoproteins. A number of studies have confirmed the role of G1b gangliosides in toxin binding. The search for the identity of the protein receptors has been far less conclusive. Recently, the nerve terminal proteins synaptotagmin I and II have been proposed as receptors for serotypes A, B and E. However, functional studies in a mammalian NMJ preparation have not been done. To more fully resolve the biochemical interactions of botulinum toxin at its target site, we propose the following specific aims. 1) examine biochemically and functionally the interactions of serotypes A, B, and E with synaptotagmins I and II at the NMJ; 2) determine the identity of NMJ proteins other than synaptotagmins I and II that bind serotypes A, B and/or E. The results of these studies will impact clinical medicine by defining the membrane targets at the NMJ that may serve as templates for the development of effective pharmacologic countermeasures to botulinum intoxication and safer toxin-like therapeutics.